The Vaccinia virion: Filling the gap between atomic and ultrastructure - Fig 8

(a) Crosslinkome for major membrane proteins A17, H3, A27, A26. Yellow, red and cyan fill: ‘Outside’, TM and ‘inside’ domains, respectively, with “Inside” and “Outside” following the convention of the program TMHMM [68] in which “inside” refers to the cytoplasmic side of plasma membrane, ER membrane or vesicles for a classically embedded TM protein, and “outside” refers to the lumenal side or the ER or external side of the plasma membrane. Vertical red arrows: AG|-specific processing sites in protein A17. Red spots (A26, A27): Cysteines that are disulfide bonded to one another. Other details as in Fig 5. (b) Arrangement, at the MV envelope, of proteins shown in panel A satisfying XL, functional and imaging data. Left side: Disulfide bonded homodimers of unprocessed A17 in the IV envelope in reticulon conformation. The unprocessed A17 N-terminus is within crosslinking range of A17’s C-terminal region. The D13 external scaffold is not depicted. Right side: Maturation of the envelope includes A17 N- and C-terminal cleavage and D13 loss. H3 is now non-canonically tail-anchored in the MV envelope with immunodominant N-terminal domain exposed, and SFEs have been added as stacked trimers of A27 and associated A26. H3’s external N-terminal domain mediates most of the interaction between A27 and the processed A17 N-terminal region, with the A17 C-terminus now out of range of H3. The H3/A26 and A27/A17 protein pairs are sufficiently proximal within the A17, H3, A27, A27 network to allow some modest direct crosslinking across their interfaces. SFEs are anchored either directly or indirectly to the core wall beneath the MV envelope by disulfide bonding (arrowed). Proteins CAHH and ATI (not depicted) are candidates for mediating such disulfides. Disulfide bonding does not involve H3 which is detergent-extractable in the absence of disulfide reduction. A27 and A26, although disulfide bonded to one another, interact tightly enough that SFEs show stability in the presence of NP40 plus disulfide reducing agent. ‘N’ and ‘C’ denote protein N- and C-termini respectively. TM protein domains are colored according to panel A and S1 Fig. Although H3 was designated ‘N-outside’ by prediction program TMHMM (opposite polarity to A17), rendering the major N-terminal domain yellow, such predictions are presumably invalid for unconventionally added (tail-anchored) TM proteins (see text). Albeit two copies of the H3-A27 trimer-A26 complex are shown to suggest SFE topology, more accurate modeling would require an understanding of protein stoichiometries.